CN105941155B - A kind of method that Manchurian ash is quickly bred using suspension culture techniques - Google Patents

Abstract

The invention discloses a method for rapid propagation of Mandshurica mandshurica by means of suspension culture technology. The seeds of Mandshurica mandshurica are selected, soaked in alcohol solution, rinsed with sterile water, put into sodium hypochlorite solution for disinfection, and then rinsed with sterile water and taken out. The mature embryos are inoculated in the seed germination medium for cultivation, and after 7-10 days, they are transferred to the liquid proliferation medium for cultivation for 15-20 days, the lateral buds are cut off, and then transferred to the liquid proliferation medium for cultivation, and the robust seedlings are domesticated for a week Then transplant into the sterilized medium until the seedlings survive. The present invention has the advantages of using the suspension culture method to induce rapid propagation of Mandshurica mandshurica, the practical method is simple, breaks the production bottleneck caused by the low germination rate of seeds, shortens the culture period, improves the reproduction coefficient, and reduces the excessive time in the tissue culture experiment process. It consumes more manpower and material resources, fundamentally improves work efficiency, saves production costs, and can achieve the purpose of industrial seedling cultivation.

Description

A kind of method that utilizes suspension culture technique to rapidly propagate Mandshurica mandshurica

technical field

The invention relates to a method for rapidly propagating mandshurica mandshurica seedlings by means of suspension culture technology, and belongs to the technical field of tree tissue culture and breeding.

Background technique

Fraxinus mandschurica Rupr. Oleaceae, Ash genus, tall deciduous tree. It takes 15 to 20 years to bloom and bear fruit under the natural growth environment, and the fruit growing period is from September to October. Mandshurica mandshurica is an ancient relic plant with a wide distribution area, but most of them are scattered. It is one of the precious trees known as the "three hard broads" in Northeast my country. The wood material is fine and the texture is beautiful. It is widely used in various fields. Due to the early development history of Mandshurica mandshurica, its natural forest resources have become less and less in recent years. Due to excessive logging, its number has been decreasing day by day. At present, not only are there few mature trees, but even the distribution area has been severely reduced. The state has adopted various measures for this endangered plant, so it is distributed in a small amount in nature reserves in individual areas.

Therefore, it has become a problem to be solved urgently to strengthen the reproduction of excellent seedlings of Mandshurica mandshurica to meet the needs of forest tree breeding. According to investigations, among the seedlings propagated by seeds, 5%-7% of super seedlings have grown more than twice the average height of the batch of seedlings. If these excellent seedlings can be multiplied and used for afforestation, it is expected to increase the growth rate of plantations. Nowadays, the breeding and cultivation technology of Mandshurica mandshurica seedlings has become a bottleneck restricting the development of breeding and its processed products, but there are few reports on Mandshurica mandshurica seedling breeding in the literature.

At present, the published research results mainly focus on the in vitro culture of immature embryos (Zhang Huijun et al., 2003), hypocotyl adventitious bud induction (Tan Yanshuang et al., 2003), axillary bud culture (Zhang Lijie et al., 2007), somatic embryos and Cytological studies on zygotic embryogenesis (Kong Dongmei et al., 2006) and tender shoot tip culture (Gu Dizhou, 2010), etc., these research results are relatively preliminary and have not yet reached the level of practical productive application. Most of the above-mentioned research materials choose axillary buds and tender stem tips, and the tree shape of Mandshurica mandshurica is tall, so the material has great limitations, and the pollution control of microorganisms inside Mandshurica mandshurica is difficult, so even if the multiplication culture is carried out, the multiplication will fail due to pollution. In Zhang Huijun's research, immature embryos were selected, but the differentiation rate was low during subculture differentiation, which did not meet the requirements for rapid propagation of seedlings in tissue culture.

Contents of the invention

The object of the present invention is to provide a kind of method for rapidly propagating Mandshurica mandshurica. The method can obviously promote the differentiation, strong seedlings and rooting of Mandshurica mandshurica, thereby providing technical support for industrialized production of Mandshurica mandshurica seedlings and the reproduction of excellent tree resources.

In order to achieve the above object, the seeds of the fine varieties of Mandshurica Fraxinus of the present invention are sterilized through explants, and their mature embryos are taken for cultivation, and then liquid suspension culture technology is used to induce plant hormones to make Mandshurica mandshurica rapidly differentiate in a short time. Strong seedlings and rooting have been established, and a system for rapid propagation of Mandshurica mandshurica by means of suspension culture technology has been established.

The invention discloses a method for rapid propagation of Mandshurica mandshurica seedlings by means of a suspension culture technique. The method adopts aseptic seedlings as suspensions for culture.

A method for rapidly propagating mandshurica mandshurica seedlings utilizing suspension culture technology, comprising the following steps:

(1) Obtain aseptic seedlings of Mandshurica mandshurica by aseptic culture technology: get the fine seedlings of Mandshurica mandshurica, soak with 70% ethanol for 30-60s in a sterile environment, then wash with water 3 times, each 1-2min; Move the seeds into 10% sodium hypochlorite solution, sterilize for 12-20 minutes, and then wash with water 5-6 times, 1-5 minutes each time; cut the top of the seeds with a sterile knife, take out the complete embryos, and inoculate them into the seed germination medium. Placed in the tissue culture room, the cultivation conditions of day and night photoperiod are as follows: the daytime light intensity is 1500-3000Lux, the light time is 12-14h/d, the temperature is 25+1°C; the nighttime temperature is 18±1°C; the seeds germinate after 7-10d, and the obtained sterile vaccine;

Among them: the seed germination medium formula is: WPM basic medium + sucrose 20-35g/L + agar 4-5g/L, pH value 5.8-6.0;

(2) Establishment of the rapid propagation method of Mandshurica mandshurica: the sterile seedlings obtained in step (1) are transferred to the rapid propagation medium, placed on a shaker for shaking culture, and the rotating speed is 100-125rpm; day and night photoperiod culture conditions It is: the daytime light intensity is 2000-3000Lux, the light time is 12-14h/d, the temperature is 25±1°C; the nighttime temperature is 18±1°C; 15-20 days, the terminal buds grow rapidly, the lateral buds differentiate, the roots are also formed, and the multiplication factor is 4 -6 times, cut off the side buds and continue to transfer to the proliferation medium;

Among them: the rapid propagation medium formula is: WPM basic medium + 6-BA 0.5-4.0mg/L + NAA 0.01-2.0mg/L + sucrose 20-35g/L, pH value 5.8-6.0;

(3) Transfer of hardened seedlings: move the robust seedlings in step (2) to the natural environment for 3 days in the tissue culture bottle, open the tissue culture bottle cap for 5 days to domesticate and train the seedlings, and then transplant them into the sterilized place after one week of domestication and hardening Treated peat soil: larch humus: vermiculite = 2:1:1, covered with a film, watered, and kept air humidity, and gradually opened the film after one week until the seedlings survived.

Compared with the existing technology, mature embryos were selected in this study, which has inherited the excellent characteristics of super seedlings. Moreover, this study uses liquid suspension culture technology, and differentiation and rooting are carried out at the same time, breaking the breakthrough that suspension culture can only cultivate cells but not culture The conclusion of the seedlings and the establishment of the suspension culture system also laid the foundation for the later use of bioreactor culture and industrial production.

Simultaneously, the present invention adopts the suspension culture method to induce the rapid propagation of Mandshurica mandshurica, and the practical technical method is simple and convenient. From explant induction to plant propagation, transplanting and seedling hardening, the whole culture cycle breaks the traditional tissue culture propagation method, and completes bud induction and rooting culture in one step. The cultivation cycle is shortened, the reproduction coefficient is improved, the excessive consumption of manpower and material resources in the tissue culture experiment process is reduced, the work efficiency is fundamentally improved, the production cost is saved, and the purpose of industrialized seedling cultivation can be achieved.

Description of drawings

Figure 1 Suspension culture of aseptic seedlings of Fraxinus mandshurica

Detailed ways

The present invention will be described in detail below in combination with specific embodiments.

The present invention adopts WPM as the base medium, and configures the combination of different plant growth substances:

Fraxinus mandshurica seed germination medium: WPM basic medium + sucrose 20/L + agar 4.5g/L, pH 5.8-6.0;

Fraxinus mandshurica rapid propagation medium: WPM basic medium + 6-BA 2.0mg/L + NAA 0.15mg/L + sucrose 20g/L, pH 5.8-6.0;

The present invention carries out according to the following steps:

(1) Take the fine seedlings of Mandshurica mandshurica, soak with 70% ethanol for 30-60s in a sterile environment, then wash with water 3 times, each time for 1-2min;

(2) Move the seeds into a 10% sodium hypochlorite solution, disinfect for 15 minutes, and then wash with water 5-6 times, each time for 1-2 minutes;

(3) Cut the top of the seed with a sterile knife, take out the complete embryo, inoculate it into the seed germination medium, and place it in the tissue culture room. 14h/d, temperature 25±1℃; night temperature 18±1℃; after 7-10d, the seeds germinated and sterile seedlings were obtained;

(4) The aseptic seedlings obtained are transferred to the rapid propagation medium, placed on a shaker for shaking culture, and the rotation speed is 120rpm; the day and night light cycle culture conditions are: daytime light intensity is 2000-3000Lux, and light time is 12-14h /d, temperature 25±1°C; night temperature 18±1°C; 15-20 days, the terminal buds grow rapidly, the lateral buds differentiate, and the roots are also formed. Cut off the lateral buds and continue to transfer them to the proliferation medium;

(5) Transfer of hardened seedlings: move the strong seedlings in the tissue culture bottle to the natural environment for 3 days, open the cap of the tissue culture bottle to domesticate the seedlings for 5 days, and transplant them into sterilized peat soil after one week of domestication and hardening : Larch humus: vermiculite = 2:1:1 medium, cover with film, water, keep air humidity, gradually open the film after one week until the seedlings survive.

The invention induces mature embryos and constructs a novel breeding system of Mandshurica mandshurica by means of suspension culture technology, breaks the production bottleneck caused by low seed germination rate, and provides conditions for large-scale production.

The above description is only a preferred embodiment of the present invention, and does not limit the present invention in any form. Any simple modifications made to the above embodiments according to the technical essence of the present invention, equivalent changes and modifications, all belong to this invention. within the scope of the technical solution of the invention.

Claims (1)

1. a kind of method that utilizes suspension culture technology to rapidly breed Mandshurica mandshurica seedlings, it is characterized in that, described method adopts Mandshurica mandshurica aseptic seedling as suspension, it comprises the steps: (1) By aseptic culture technology, using mature embryos of excellent seedlings of Mandshurica mandshurica as material, inducing to obtain aseptic seedlings of Mandshurica mandshurica; (2) Put the aseptic seedlings of Mandshurica mandshurica obtained in step (1) in the liquid proliferation medium, the liquid medium is WPM basic medium+6-BA 0.5-4.0mg/L+NAA 0.01-2.0mg/L+sucrose 20-35g/L, pH 5.8-6.0; (3) Place the inoculated culture bottle on a shaking table for shaking culture, the rotation speed is 100-125rpm, the light intensity is 2000-3000Lux, the light time is 12-14h/d, the temperature is 25±1°C, and cultured for 15-20 days, it can be The terminal buds grow rapidly, the lateral buds differentiate, and the roots are also formed. The terminal buds and lateral buds are cut off, and continue to be transferred to the liquid proliferation medium for cultivation; (4) The aseptic seedlings obtained in the step (3) are subjected to hardening and transplanting to obtain excellent seedlings of Fraxinus mandshurica.